Focal adhesions (FAs) are sites where cells in culture adhere tightly to the underlying extracellular matrix (ECM). They serve to anchor bundles of actin filaments to the plasma membrane and are sites of adhesion-mediated cell signaling, implicated in growth control. They are particularly rich in phosphotyrosine. This grant is aimed at understanding how FAs assemble, at determining the role of tyrosine phosphatases (PTPases) in FAs and at exploring the consequences of the interaction between the FA protein vinculin and the lipid PIP2. Activation of the GTP-binding protein Rho stimulates assembly of FAs. Determining the pathway from Rho to FA assembly is a major goal of this proposal. Following our discovery that Rho-stimulated contractility drives the formation of FAs, we will determine whether constitutively active myosin light chain (MLC) kinase or inhibitors of myosin phosphatase (MPP) induce contractility and FA assembly. We will test the hypothesis that Rho activates a kinase, such as p160ROCK or PKN, that phosphorylates and inhibits MPP and thereby elevates MLC phosphorylation. Dominant negative mutants of Rho- activated kinases will be used to explore this pathway. Mutant Rho constructs will be generated to define the Rho-activated kinase(s) that is responsible for stimulating contractility. The activation of Rho in response to integrin-mediated adhesion will be explored. Using dominant negative mutants of CDC42 and Rac, we will test the hypothesis that adhesion to ECM stimulates a cascade involving successively CDC42, Rac and Rho. The PTPases that regulate FA tyrosine phosphorylation will be examined. We will attempt to identify and characterize the PTPase that we have detected in association with integrins. We will explore the consequences of overexpressing or inhibiting this PTPase. Finally, we will determine whether changes in vinculin expression affect anchorage-dependent growth by modulating PIP2 availability. The effect of vinculin on PIP2 hydrolysis will be studied. We will determine which domain of vinculin restores anchorage-dependent growth when transfected into cells with reduced vinculin expression.